ABSTRACT Our long-term goal is to elucidate the molecular mechanisms governing the restructuring of the genome that allows for the transition from a specified cell type to a totipotent state. This transition occurs rapidly and efficiently immediately following fertilization, during the initial stages of embryonic development. At this critical developmental stage, the zygotic genome is transcriptionally quiescent. Only when the cells have become totipotent and poised to differentiate does widespread zygotic transcription initiate. Despite the fact that this delayed transcriptional activation of zygotic genome is nearly universal among metazoans, the mechanisms governing this process and how it relates to the coordinated establishment of a totipotent state remain unknown. We have recently demonstrated that the Drosophila protein Zelda (ZLD) acts globally to facilitate the activation of the zygotic genome and propose that it is doing so by establishing a permissive chromatin environment. Through genome-wide binding studies, we have shown that ZLD is bound early in development to thousands of genomic regions that subsequently drive the first wave of zygotic transcription. These loci are later defined as regions of open chromatin and are bound by a large number of transcription factors that govern embryonic patterning. Through our initial studies, we are well positioned to define the mechanisms by which the zygotic genome is remodeled to create an environment permissive to transcriptional activation and cellular differentiation. We will use genetic, genomic, and biochemical strategies to determine the mechanism by which ZLD is acting as a pioneer factor to shape the chromatin environment of the early embryo by: 1) determining the role of ZLD in organizing the chromatin structure of the early embryo and 2) identifying cofactors required for ZLD-mediated genome activation. The mechanistic insights we will gain into ZLD function will advance our understanding of how totipotent genomes are established generally. Ultimately this work will have important implications for the generation of totipotent mammalian stem cells both in culture and in vivo.